Hoxa9 is a homeodomain containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. More than 50% of acute myeloid leukemia (AML) cases show overexpression of Hoxa9, almost always in association with overexpression of its cofactor Meis1. In a study of gene expression in human AMLs, high expression HOXA9 was the single most predictive mark for poor prognosis. A wide range of data suggests that Hoxa9 and Meis1 play a synergistic causative role in AML, though the molecular mechanisms leading to transformation by Hoxa9 and Meis1 remain elusive. Understanding Hoxa9-mediated leukemogenesis first requires a better understanding of what confers binding specificity of Hox family proteins. All Hox proteins bind the ubiquitous TAAT motif through their highly homologous homeodomains, which alone cannot account for their tight control of transcriptional activity. Additional sequence specificity is achieved in vivo through association with other DNA-binding cofactors, such as Meis1. Another level of regulation is likely conferred by diverse sets of collaborators that direct Hox protein specificity, but the identity of these proteins and the mechanisms through which they regulate Hox binding have yet to be elucidated. We have made considerable progress towards determining potential collaborators by characterizing in vivo binding sites of Hoxa9 and Meis1 and by identifying proteins that interact with the Hoxa9 complex. We found that Hoxa9 and Meis1 bind to evolutionarily conserved sites that contain an epigenetic signature consistent with enhancer sequences. De novo motif analysis of the binding regions showed a marked enrichment of motifs for transcription factors (TF) in the C/EBP, ETS, and STAT families. Subsequent mass spectrometry and coimmunoprecipitation experiments have confirmed association of the Hoxa9 complex with C/ebp a and Stat5. We have recently generated leukemic cell line model systems that conditionally express C/ebp a, Pu.1 and Stat5, and we will use these cells to study the role of each protein in targeting Hoxa9 to specific binding sites in myeloid and lymphoid lineages. We will also use these models to determine the effect of C/ebp a, Pu.1 and Stat5 on the transcriptional activity of Hoxa9. Finally, we will use a cell line we have established with conditional expression of the chromatin remodeler, Brg1, to determine the functional relationship between Brg1 and C/ebp a in modulating Hoxa9 complex activity. Together these studies will provide many new mechanistic insights into leukemogenesis mediated by high-level Hoxa9 expression. )